FIG 7 .

EspC translocation inside epithelial cells is blocked by anti-EspB/anti-EspD antibodies. (A) EspC is not detected in the cytoplasmic fraction by using anti-EspB/anti-EspD antibodies. HEp-2 cells were infected with EPEC ΔespC for 1 h and incubated with either anti-EspB/anti-EspD or anti-Pic antibodies for 45 min; finally, exogenous EspC (0.9 μM) was added to the cells and incubated for 2 h. Cytoplasm fractions of infected cells were analyzed by Western blotting using either anti-EspC or anti-EspF antibodies, and actin detection was used as a loading control. An image representative of results of four independent experiments is shown. (B) EspC internalization is inhibited by blocking the EspB/EspD pore. HEp-2 cells were infected and treated with the antibodies and exogenous EspC as described for panel A. In the first panel row, the cells were not permeabilized and were processed for confocal microscopy for decorating the EspB/EspD pores where the bacteria were interacting with the epithelial cells. EspC internalization was detected in cells permeabilized and processed for confocal microscopy. EspC was detected with anti-EspC antibodies followed by anti-mouse antibodies conjugated to fluorescein isothiocyanate (FITC; green). EspB/EspD proteins were detected with anti-EspB and anti-EspD antibodies, respectively, followed by TRITC goat anti-rabbit IgG (red). Actin cytoskeleton was detected with rhodamine-phalloidin (red) and DNA with TO-PRO-3 iodide (blue). Representative images from three independent experiments are shown. The white squares are the zoom panels. Bar, 5 μm.